Antibacterial compounds and process for production thereof

ABSTRACT

The antibacterial compound S551-II (Reductiomycin) is produced by culturing a microorganism belonging to the genus Streptomyces. 
     S551-II-A is prepared by sublimation of the compound S551-II.

SUMMARY OF THE INVENTION

The present invention relates to new antibacterial compounds S551-II (Reductiomycin) and S551-II-A. The compound S551-II is produced by culturing a microorganism belonging to the genus Streptomyces in a nutrient medium. The compound S551-II-A is produced by sublimating the compound S551-II.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the infrared absorption spectrum of the compound S551-II.

FIG. 2 shows the infrared absorption spectrum of the compound S551-II-A.

FIG. 3 shows the nuclear magnetic resonance spectrum of the compound S551-II.

DETAILED EXPLANATION OF THE INVENTION

The present invention relates to new antibacterial compounds represented by the following general formula: ##STR1## wherein R is ##STR2## (the compound having the following formula ##STR3## is referred to as Compound S551-II hereinafter) or ##STR4## (the compound having the following formula ##STR5## is referred to as S551-II-A hereinafter).

The compound S551-II is also designated as Reductiomycin.

The anitbacterial compound S551-II is produced by culturing a microorganism belonging to the genus Streptomyces and being capable of producing the anitbacterial compound in a nutrient medium. The antibacterial compound is accumulated in the culture liquor and is isolated therefrom.

The anitbacterial compound S551-II-A is produced by sublimation of the compound S551-II.

The present compound S551-II has the following physicochemical properties.

Appearance: A yellow fine crystal

Melting point: 215° C. (decompose)

[α]_(D) ²³° : +281° (c=0.30, acetone)

Molecular weight: 293 (by the mass spectrometry)

Elementary analysis: C: 57.36%, H: 5,25%, N: 4.73%

Molecular formula: C₁₄ H₁₅ O₆ N

Ultraviolet absorption spectrum (the maximum value, ε value):

273 nm (28655) (in methanol)

288 nm (21600) (in 0.1 N HCl-methanol)

Color reactions:

Enrlich's reation (in HCl): positive

Ferric chloride reaction: positive

Nitropurusside reaction (in alkaline): positive

2,4-Dinitrophenylhydrazine reaction: positive

Pine-shaving reaction: negative

Ninhydrin reaction: negative

Schiff's reaction: negative

Sakaguchi's reaction: negative

Infrared absorption spectrum by the paste method is illustrated in FIG. 1.

PMR spectrum measured in CDCl₃ by usng TMS as an internal standard is illustrated in FIG. 3.

Based on the foregoing data, the compound S551-II is considered to have the structural formula shown earlier.

The compound S551-II-A has the following physicochemical properties.

Appearance: A yellowish crystal

Melting point: 215° C. (sublimate)

[α]_(D) ²³° : 0° (c=0.30, dimethylsulfoxide)

Molecular weight: 233 (by the mass spectrometry)

Elementary analysis: C: 60.98%, H: 4.59%, N: 6.06%

Molecular formula: C₁₂ H₁₁ O₄ N

Ultraviolet absorption spectrum (the maximum value, ε value): 260 nm (52900) (in methanol and 0.1 N-NaOH).

Color reactions:

Ehrlich's reaction (in HCl): positive

Ferric chloride reaction: positive

2,4-dinitrophenylhydrazine reaction: positive

Ninhydrin reaction: negative

Schiff's reaction: negative

Sakaguchi's reaction: negative

Nitroprusside reaction: negative

Solubilities: The compound is soluble in acetone, dimethylsulfoxide and alkaline solution, slightly soluble in methanol and insoluble in benzene, chloroform ethylether and hexane.

Infrared absorption spectrum of S551-II-A by the paste method is illustrated in FIG. 2.

Based on the foregoing data, the compound S551-II-A is considered to have the structural formula shown earlier.

Now, the process for producing S551-II is described below.

The new antibacterial compound S551-II is produced by culturing a microorganism belonging to the genus Streptomyces and being capable of producing the compound. A suitable microorganism belongs to Streptomyces griseorubiginosus. Its typical strain is Streptomyces griseorubiginosus KY 11448 (FERM-P 3836) (NRRL 11,268).

The strain has the following properties.

I. Morphology

The spore forming mycelium shows flexous simple branching. A chain of ten or more spores is formed.

The surface of the spore is smooth and the diameter of its minor axis and major axis are 1.0 μ and 2.5 μ, respectively. The sporophore is formed on the aerial mycelium.

II. Culture characteristics

    ______________________________________                                                          Color of the                                                                   substrate mycelium                                                                             The                                                            Aerial    The   reverse                                                                              Soluble                                 Medium  Growth   Mycelium  surface                                                                              side  pigment                                 ______________________________________                                         Sucrose-                                                                               Poor     Poor      White White                                         nitrate Flat     White(a)  (a)   (a)   None                                    agar                                                                           Glucose-                                                                               Moderate Moderate  Cream Light                                         asparagine                                                                             Raised   Light     (11/2a)                                                                              Yellow                                                                               None                                    agar             Ivory           (11/2ea)                                                       (2ca)                                                         Glycerin-                                                                              Moderate Moderate  Cream Light                                         asparagine                                                                             Flat     Light     (11/2a)                                                                              yellow                                                                               None                                    agar             Ivory           (11/2ea)                                                       (2ca)                                                         Starch  Good     Good      Cream Light Butter                                  agar    Raised   Cream     (11/2a)                                                                              Yellow                                                                               Yellow                                                   (11/2a)         (11/2ea)                                                                             (11/2ga)                                                                 Must-                                         Tyrosine                                                                               Good     Good      Dull  ard   Antique                                 agar    Raised   Cream     Gold  Brown Gold,                                                    (11/2a)   (2ng) (2ni) (11/2ne)                                Nutrient                                                                               Good               Bright                                                                               Bright                                                                               Amber                                   agar    Raised   None      Gold  Gold  (3pc)                                                              (2nc) (2nc)                                         Yeast   Good     Good      Golden                                                                               Golden                                                                               Mustard                                 extract-                                                                               Raised   Putty     Brown Brown Gold                                    malt                                                                           exract           (11/2ec)  (3pg) (3pg) (2ne)                                   agar                                                                           Oatmeal Poor               Dull  Dull  Clove                                   agar    Raised   None      Gold  Gold  Brown                                                              (2ng) (2ng) (3ni)                                   ______________________________________                                    

The color indications are give according to the classifications in the Color Harmony Manual (Container Corporation of America).

III. Physiological properties

Growth temperature: 25 to 38° C.

Liquefaction of gelatin: negative

Hydrolysis of starch: positive

Coagulation and peptonization of skin milk: negative

Liquefaction of skim milk: positive

Formation of melanoid pigments: positive

IV. Utilization of carbon sources

    ______________________________________                                         Carbon source        Utilization                                               ______________________________________                                         D-Arabinose          --                                                        D-Xylose             --                                                        D-Glucose            2+                                                        D-Fructose           2+                                                        Sucrose              --                                                        Inositol             --                                                        L-Rhamnose           --                                                        Raffinose            +                                                         D-Mannitol           2+                                                        ______________________________________                                    

On the basis of the above observations and the description of E. Kuster, International Journal of Systematic Bacteriology 22 (3), 139 (1972), the strain is identified as Streptomyces griseorubiginosus.

As the fermentation medium employed in the present process, any synthetic or natural medium can be employed, so long as it contains a proper carbon source, a nitrogen source, inorganic materials and other nutrients necessary for the growth of the microorganism.

As the carbon source, various carbohydrates such as glucose, fructose, sucrose, gallactose, xylose, sorbitol, mannitol, glycerol, starch, starch hydrolyzate liquor, molasses, blackstrap molasses, etc., various hydrocarbons such as ethane, propane, butane, n-paraffine, kerosene, etc., various organic acids such as acetic acid, fumaric acid, succinic acid, lactic acid, pyruvic acid and alcohols such as methanol, ethanol, etc. may be used.

As the nitrogen source, aqueous ammonia, various inorganic and organic ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium acetate, etc., urea, acid amides, amines, amino acids, defatted cotton seed, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, fish meal or its digested product, defatted soybean or its digested product, soybean protein hydrolyzate; various microbial cells or its digested product, etc. may be used.

As the inorganic materials, dipotassium monohydrogen phosphate, monopotassium, dihydrogen phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium carbonate, magnesium phosphate, etc. may be used.

If other nutrients are necessary for the growth of the microorganisms used in the present invention, they must, of course, be present in the medium. In some cases, these nutrients are added as components of the natural substances in the medium such as the organic nitrogen sources mentioned above.

Culturing is carried out under aerobic conditions such as with shaking or aeration-agitation. Suitable culturing temperature is usually 23° to 38° C. It is desirable to keep the pH of the medium at 3 to 8, preferably around neutrality throughout culturing.

Culturing is usually carried out for 1 to 7 days to accumulate the compound S551-II in the culture liquor.

After completion of the culturing, microbial cells and precipitates are removed from the culture liquor by filtration or centrifugation and the compound S551-II may be recovered and isolated from the resultant solution by combination of ion exchange treatment, column chromatography using silica gel, etc.

The compound S551-II-A is prepared as follows. The compound S551-II is heated at a temperature of 215° or more and is sublimated accompanying with deacetic acid reaction to form compound S551-II-A. The product crystallized on the glass plate was collected and washed with methanol to obtain a pure S551-II-A as crystals.

The antibacterial activity of the compound S551-II against various microorganisms by disc method (pH: no adjustment) is shown in the following Table 1.

                  Table 1                                                          ______________________________________                                         Microorganism tested     MIC (mcg/ml)                                          ______________________________________                                         Aspergillus niger IAM 2026                                                                              250                                                   Aspergillus niger ATCC 6275                                                                             1,000                                                 Aspergillus oryzae NRRL 692                                                                             62.5                                                  Penicillium chrysogenum IAM 7142                                                                        125                                                   Penicillium chrysogenum Q 176                                                                           62.5                                                  Mucor spinescens IAM 6071                                                                               500                                                   Dusariium moniliforme IAM 5062                                                                          125                                                   Myrothecium verrucaria IAM 5063                                                                         125                                                   Trycophyton mentagrophytes IAM 5064                                                                     4                                                     Irycoderma T-1 ATCC 9645 16                                                    Chaetomium globosum ATCC 6025                                                                           500                                                   gypseum IFO 5948         125                                                   Alternaria solani IFO 5924                                                                              8                                                     Cladosporium herbarum Link Fr IAM 5059                                                                  31.2                                                  Bacillus subtilis PCI 219                                                                               62.5                                                  Bacillus subtilis IAM 1026                                                                              31.2                                                  Bacillus subtilis NA 64  62.5                                                  Sarcina lutea IAM 1099   125                                                   Staphylococcus aureus FAD 209P                                                                          62.5                                                  Diplococcus pneumoniae   1000                                                  Escherichia coli IAM 1268                                                                               1000                                                  Escherichia coli ATCC 3655                                                                              500                                                   Serratia marcescens IAM 1022                                                                            >1000                                                 Proteus vulgaris HX19 IAM 1025                                                                          >1000                                                 Pseudomonas aeruginosa IAM 1156                                                                         >1000                                                 Pseudomonas fluorescens ATCC 27                                                                         >1000                                                 Salmonella enteritidis   >1000                                                 Shigella sonnei E23      >100                                                  Klebsiella pneumoniae 348                                                                               >1000                                                 Candida albicans IAM 4888                                                                               >1000                                                 Saccharomyces cerevisiae IAM 4485                                                                       1000                                                  Saccharomyces rouxii M-9 100                                                   ______________________________________                                    

The antibacterial activity of the compound S551-II-A against various microorganism by agar dilution method (pH 7.0) is shown in Table 2.

                  Table 2                                                          ______________________________________                                         Microorganism tested    MIC (mcg/ml)                                           ______________________________________                                         Bacilus subtilis ATCC 10707                                                                            200                                                    Staphylococcus aureus ATCC 6538P                                                                       200                                                    Klebsiella pneumoniae ATCC 10031                                                                       200                                                    Proteus vulgaris ATCC 6897                                                                              25                                                    Escherichia coli ATCC 3655                                                                             500                                                    ______________________________________                                    

As it is obvious the above description, the compounds of the present invention are useful to clean and disinfect laboratory glassware and surgical instruments, and may also be used in combination with various soaps for sanitation purposes and in cleaning and sanitizing hospital rooms and areas used for the preparation of food.

Practice of certain specific embodiments of the invention is illustrated by the following representative examples.

EXAMPLE 1

Composition of fermentation medium (as same as seed medium)

    ______________________________________                                         Soluble starch        2%                                                       Gluten meal           2%                                                       Defatted cotton seed  2%                                                       Dry yeast             2%                                                       CaCO.sub.3            0.3%                                                     MgSO.sub.4.7H.sub.2 O 0.07%                                                    Silicon KS66          0.1% (V/V)                                               (antifoaming agent)                                                            ______________________________________                                    

Streptomyces griseorubiginosus KY 11448 (FERM-P No. 3836) (NRRL 11,268) is used as a seed strain.

200 ml of seed culture obtained by culturing the above seed strain in 300 ml of seed medium in a Sakaguchi-flask for 50 hrs in advance is transferred into 30 l-Jar containing 15 l of said fermentation medium. Culturing is carried out at 30° C. for 72 hrs with aeration of b 14 l/min. and stirring at 300 r.p.m.

After culturing, culture broth is filtrated to remove microbial cells and the filtrate is extracted with about 15 l of ethylacetate. The resulting extract is concentrated to dryness under reduced pressure. The above separated microbial cells is extracted with acetone and the resulting extract is concentrated to dryness under reduced pressure. Then, the obtained residue is extracted with ethylacetate and concentrated to dryness under reduced pressure. The resulting two residues are collected and is subjected to chromatography using silica gel (200 ml by volume) and then washed with 500 ml of benzene. Then, elution is carried out with each of 500 ml of benzene-methanol (1% by volume), benzene-methanol (2% by volume) and benzene-methanol (3% by volume). The eluates by benzene-methanol are collected and concentrated to dryness and then the resulting residue is washed with about 30 ml of ethylether-chloroform (10:1 by volume). Then, recrystallization is carried out twice from about 8 ml of chloroform and about 5 ml of acetone, whereby 30 mg of compound S551-II is obtained as yellow fine crystal.

EXAMPLE 2 Synthesis of compound S551-II-A

Compound S551-II obtained in Example 1 is put on heater and is covered with Petri dish. Then, the compound is heated at 215° C. whereby compound S551-II is sublimated and the volatile component is adhered to wall of Petri dish to form crystals. The resulting crystals are collected and are washed with methanol, whereby pure compound S551-II-A is obtained. 

What is claimed is:
 1. A process for producing compound S551-II represented by the formula ##STR6## which comprises culturing a microorganism having the identifying characteristics of Streptomyces griseorubiginosus FERM-P No. 3836, NRRL 11,268 in a nutrient medium until said compound is accumulated in the culture liquor and recovering said compound from said culture liquor.
 2. The process according to claim 1 wherein said culturing is carried out at a temperature of from 23° to 38° C. 